1. Field of the Invention
This invention relates to a method of making antihemophilic factor (AHF, Factor VIII) concentrate which is substantially fibrinogen free. AHF is a blood phasma protein useful for therapeutic administration to patients having hemophilia.
Hemostatis, the biochemistry of blood coagulation, is an extremely complex and as yet not completely understood phenomena whereby normal whole blood and body tissue prevent an excess loss of blood from a ruptured blood vessel. The total mechanism of blood coagulation is affected through the coordinated interaction of biochemical substances contained in three basic physiologic systems, namely, extravascular tissue such as subcutaneous tissue, muscle tissue, and skin; the blood vessel wall; and intravascular components, including blood plasma proteins, blood plasma factors, and platelets. By far the most important, and yet least understood, of the biochemical considerations affecting clotting, involve the intervascular blood components.
Since most of the blood clotting diseases in man occur in the intervascular blood system, usually due to a deficiency or inactivation of one or more blood plasma factors, great effort has been expanded in this direction by scientific research in an attempt to understand the role blood plasma factors play in the biochemical mechanism of blood clotting. Although in recent years much progress has been made in understanding the complexities of blood clotting, many more years of painstaking effort will be required before man finally gains sufficient knowledge to effectively ameliorate blood clotting disease. In the meantime, the state of the art with respect to the treatment of most blood clotting diseases will continue to be the administration of therapeutic pharmaceutical and biochemical substances in an attempt to relieve the adverse effects of these diseases.
A great deal of medical research into blood clotting diseases has been directed towards finding an acceptable treatment for hemophilia, a genetically induced disease characterized by the loss of clotability of otherwise normal whole blood. Although the precise cause of hemophilia is not known, one of the most popular theories suggests that it may be because of the absence of or a greatly inhibited presence of the active form of AHF in otherwise normal plasma from whole blood. At present, although hemophilia cannot be cured, it can often be treated therapeutically be the administration of AHF to an AHF-deficient individual.
2. Description of the Prior Art
AHF derived from blood obtained from a normal and healthy donor is administered either by the transfusion of whole blood or blood plasma, or by the infusion of AHF plasma protein concentrate which has been extracted from the plasma of normal human whole blood. However, these techniques have often proved therapeutically unsatisfactory as will hereinafter appear.
When whole blood or blood plasma transfusions are used to relieve a hemophiliac, one must exercise great care to select reasonably fresh blood or plasma because the biologic activity of AHF is extremely labile upon storage under normal conditions. Even laboratory techniques, such as lyophilization and cryogenic preservation, will not prevent substantial loss of biologic activity of AHF over time. Another major disadvantage of whole blood or blood plasma transfusions is that the can introduce unwanted proteinaceous and non-proteinaceous material in the recipient's blood stream, often causing allergic reactions to sensitive patients, viral infections such as hepatitis, or hypervolumetric reactions to those persons who require extensive amounts of AHF to initiate clotting.
The third therapeutic technique; namely, i.v. administration of AHF plasma concentrates is presently being used extensively. These concentrates are being developed primarily to circumvent the aforementioned troublesome and often times dangerous side effects caused by whole blood or blood plasma transfusions. Essentially, AHF plasma concentrate might be characterized as AHF-rich blood plasma extracts from which some blood plasma proteins, such as the gamma globulins, most other blood plasma factors, and many inorganic chemicals have been removed. However, even currently available AHF-rich blood plasma concentrates may contain impurities which can cause deleterious effects when administered to man so that a need for a purer, more therapeutically acceptable AHF plasma concentrate still exists.
Of particular importance in the development of a more therapeutically acceptable AHF product has been the research directed towards the removal of fibrinogen from AHF plasma concentrate. Fibrinogen, contained in an AHF product, is an especially intolerable impurity because of its tendency to interfere with the blood platelets function of releasing essential clotting factors into the patient's blood stream. Although the exact mechanism has not been conclusively determined, it now seems that the fibrinogen coats the cellular membrane of the platelet and inhibits the passage of the clotting factors from the platelet through its membrane into the blood plasma.
Another disadvantage arising from the presence of fibrinogen impurities in an AHF plasma concentrate is the tendency of fibrinogen to develop strong antigenic rejection responses in many patients who have been subjected to repeated and prolonged fibrinogen-rich AHF plasma concentrate infusions. It has also been medically shown that repeated massive doses of fibrinogen contained in an AHF plasma concentrate can cause the same antigenetic response of the patient to become sensitive to other proteins in the AHF plasma concentrate, such as AHF, which might not normally be rejected if administered separately. Once anti-AHF antigens are formed within a patient, further therapeutic administration of AHF becomes less beneficial.
Because of the similar physical and chemical properties of AHF and fibrinogen, standard proteinaceous separation techniques, such as electrophorisis, chromatography, and solubility differentials, have not been able to effect a sharp separation of the two proteins to produce a therapeutically acceptable fibrinogen-free AHF product.
Accordingly, the need exists for a process whereby a fibrinogen-free AHF plasma concentrate of high biologic activity might be derived from a biological sample containing a high concentration of fibrinogen.
Accordingly, it is the prime objective of this invention to provide a therapeutically acceptable AHF product which is substantially free of fibrinogen.
Another object of this invention is to provide a method of producing a substantially fibrinogen-free AHF concentrate from a cryoprecipitate suspension containing as its major components fibrinogen and AHF without substantial loss of AHF biologic activity.
Still another object of this invention is to provide an AHF concentrate useful for administration to man which will not cause damage to body tissue or organs.
A still further object of this invention is to provide a process whereby a therapeutically acceptable AHF product can be made more inexpensively and simply than was heretofore possible.
These and still further objects as shall hereinafter appear are fulfilled by the present invention in a remarkably unexpected fashion as will be readily discerned from the following detailed description of certain exemplary embodiments of this invention, especially when read in conjunction with the accompanying drawing showing a flow diagram of a process embodying the present invention.
As used herein, the term cryoprecipitate defines the solid phase fraction of human plasma obtained as a result of thawing frozen human plasma at 0.degree. C..+-.1.degree. C. and removing the liquid phase. This solid fraction contains as its primary components AHF, fibrinogen, and various cold insoluble globulin proteins.
The present invention is predicated upon the discovery of a process whereby AHF can be substantially extracted from the fibrinogen and cold insoluble components of the cryoprecipitate without significant loss of AHF biologic activity.
More particularly, this invention is a process whereby the fibrinogen component in a cryoprecipitate is selectively denatured and extracted from the AHF component of the cryoprecipitate by a rapid heating and cooling step. Upon removal of the denatured fibrinogen component of the cryoprecipitate by precipitation, the resulting supernatant contains a therapeutic quality, substantially fibrinogen-free AHF product of high AHF biologic activity.
Our invention is based on the fact that each individual protein molecular retains its biological activity or capacity to function only within specific upper and lower temperature limits. When the temperature of the protein surpasses that upper temperature limit, it begins to change its three dimensional steriochemical configuration. When the change becomes permanent and all biological activity is lost, the protein is said to be denatured.